The question of whether endogenous prolactin might be detectable in normally lactating rat mammary glands (LMG) was examined by light microscopic immunohistochemistry. In an initial study, sections of LMG were incubated sequentially with rabbit anti-rat prolactin (APRL), [I]; anti-rabbit .gamma.-globulin:peroxidase conjugate (Ar.gamma.G-per), prepared from goat anti-rabbit .gamma.-globulin and horseradish peroxidase; and 3,3''-diaminobenzidine mixed with H2O2(DAB). Use of this 3-step procedure led to staining of stromal, parenchymal and intraluminal elements. Control procedures which omitted step I produced similar staining, caused by direct binding of the Ar.gamma.G-per to endogenous tissue components (possibly rat immunoglobulins). This precluded detection of indirect, APRL-linked, binding of AR.gamma.G-per. Direct binding of Ar.gamma.G-per was prevented by preincubating LMG sections with goat anti-monkey .gamma.-globulin (AM.gamma.G), following which, the 3-step reaction sequence (APRL, AR.gamma.G-per and DAB) led to APRL-dependent staining, both within the alveolar lumina and in the apices of epithelial cells. This staining, presumably of endogenous prolactin, appeared to be specific, since it did not occur when absorbed APRL (APRL admixed with highly purified rat prolactin) was substituted for APRL in step I of the reaction sequence. When an incubation with purified rat prolactin was interposed between the AM.gamma.G pretreatment and the 3-step reaction sequence, staining of epithelial elements was intensified, indicating that these cells had contained unoccupied prolactin-binding sites. The apparent presence of both prolactin-binding sites and endogenous immunoreactive prolactin in LMG alveolar cells strongly suggests that this protein hormone can enter these target cells under physiological conditions.